Icos gene disruption in non-obese diabetic mice elicits myositis associated with anti-troponin T3 autoantibodies.

AIMS: Idiopathic inflammatory myopathies (IIM) are autoimmune inflammatory disorders leading to skeletal muscle weakness and disability. The pathophysiology of IIM is poorly understood due to the scarcity of animal disease models. Genetic deletion of Icos or Icosl (inducible T cell co-stimulator/ligand) in non-obese diabetic (NOD) mice leads to muscle disease. Our aim was to characterise Icos-/- NOD myopathy and to search for novel autoantibodies (aAbs) in this model. METHODS: Diabetes, weight, myopathy incidence/clinical score and grip strength were assessed over time. Locomotor activity was analysed with the Catwalk XT gait analysis system. Muscle histology was evaluated in haematoxylin/eosin and Sirius red-stained sections, and immune infiltrates were characterised by immunofluorescence and flow cytometry. 2D gel electrophoresis of muscle protein extracts and mass spectrometry were used to identify novel aAbs. NOD mice were immunised with troponin T3 (TNNT3) in incomplete Freund's adjuvant (IFA) and R848. An addressable laser bead immunoassay (ALBIA) was developed to measure aAb IgG serum levels. RESULTS: Icos-/- NOD mice did not exhibit diabetes but developed spontaneous progressive myositis with decreased muscle strength and altered locomotor activity. Muscle from these mice exhibited myofibre necrosis, myophagocytosis, central nuclei, fibrosis and perimysial and endomysial cell infiltrates with macrophages and T cells. We identified anti-TNNT3 aAbs in diseased mice. Immunisation of NOD mice with murine TNNT3 protein led to myositis development, supporting its pathophysiological role. CONCLUSIONS: These data show that Icos-/- NOD mice represent a spontaneous model of myositis and the discovery of anti-TNNT3 aAb suggests a new autoantigen in this model.

LasB and CbpD Virulence Factors of Pseudomonas aeruginosa Carry Multiple Post-Translational Modifications on Their Lysine Residues.

Pseudomonas aeruginosa is a multi-drug resistant human pathogen largely involved in nosocomial infections. Today, effective antibacterial agents are lacking. Exploring the bacterial physiology at the post-translational modifications (PTM) level may contribute to the renewal of fighting strategies. Indeed, some correlations between PTMs and the bacterial virulence, adaptation, and resistance have been shown. In a previous study performed in P. aeruginosa, we reported that many virulence factors like chitin-binding protein CbpD and elastase LasB were multiphosphorylated. Besides phosphorylation, other PTMs, like those occurring on lysine, seem to play key roles in bacteria. In the present study, we investigated for the first time the lysine succinylome and acetylome of the extracellular compartment of P. aeruginosa by using a two-dimensional immunoaffinity approach. Some virulence factors were identified as multimodified on lysine residues, among them, LasB and CbpD. Lysine can be modified by a wide range of chemical groups. In order to check the presence of other chemical groups on modified lysines identified on LasB and CbpD, we used 1- and 2- dimensional gel electrophoresis approaches to target lysine modified by 7 other modifications: butyrylation, crotonylation, dimethylation, malonylation, methylation, propionylation, and trimethylation. We showed that some lysines of these two virulence factors were modified by these 9 different PTMs. Interestingly, we found that the PTMs recovered on these two virulence factors were different than those previously reported in the intracellular compartment.

Neuroprotective effect of grape seed extract on brain ischemia: a proteomic approach.

Stroke is one of the leading causes of long-lasting disability in human and oxidative stress an important underlying cause. Molecular insights into pathophysiology of ischemic stroke are still obscure, and the present study investigated the protective effect of high dosage Grape Seed Extract (GSE 2.5 g/kg) on brain ischemia-reperfusion (I/R) injury using a proteomic approach. Ischemia was realized by occlusion of the common carotid arteries for 30 min followed by 1 h reperfusion on control or GSE pre-treated rats, and a label-free quantification followed by mass spectrometry analysis used to evaluate I/R induced alterations in protein abundance and metabolic pathways as well as the protection afforded by GSE. I/R-induced whole brain ionogram dyshomeostasis, ultrastructural alterations, as well as inflammation into hippocampal dentate gyrus area, which were evaluated using ICP-OES, transmission electron microscopy and immuno-histochemistry respectively. I/R altered the whole brain proteome abundance among which 108 proteins were significantly modified (35 up and 73 down-regulated proteins). Eighty-four proteins were protected upon GSE treatment among which 27 were up and 57 down-regulated proteins, suggesting a potent protective effect of GSE close to 78%of the disturbed proteome. Furthermore, GSE efficiently prevented the brain from I/R-induced ion dyshomeostasis, ultrastructural alterations, inflammatory biomarkers as CD56 or CD68 and calcium burst within the hippocampus. To conclude, a potent protective effect of GSE on brain ischemia is evidenced and clinical trials using high dosage GSE should be envisaged on people at high risk for stroke.

Lysine Succinylation and Acetylation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a multi-drug-resistant human opportunistic pathogen largely involved in nosocomial infections. Unfortunately, effective antibacterial agents are lacking. Exploring its physiology at the post-translational modifications (PTMs) level may contribute to the renewal of combat tactics. Recently, lysine succinylation was discovered in bacteria and seems to be an interesting PTM. We present the first succinylome and acetylome of P. aeruginosa PA14 cultured in the presence of four different carbon sources using a 2D immunoaffinity approach coupled to nanoliquid chromatography tandem mass spectrometry. A total of 1520 succinylated (612 proteins) and 1102 acetylated (522 proteins) lysine residues were characterized. Citrate was the carbon source in which we identified the higher number of modified proteins. Interestingly, 622 lysine residues (312 proteins) were observed either acetylated or succinylated. Some of these proteins, were involved in virulence, adaptation, resistance, and so on. A label-free quantification points out the existence of different protein forms for a same protein (unmodified, succinylated or acetylated) and suggests different abundance as a function of the carbon sources. This work is a promising starting point for further investigations on the biological role of lysine succinylation in P. aeruginosa.

Impaired vitreous composition and retinal pigment epithelium function in the FoxG1::LRP2 myopic mice.

High myopia (HM) is one of the main causes of visual impairment and blindness all over the world and an unsolved medical problem. Persons with HM are predisposed to other eye pathologies such as retinal detachment, myopic retinopathy or glaucomatous optic neuropathy, complications that may at least partly result from the extensive liquefaction of the myopic vitreous gel. To identify the involvement of the liquid vitreous in the pathogenesis of HM we here analyzed the vitreous of the recently described highly myopic low density lipoprotein receptor-related protein 2 (Lrp2)-deficient eyes. Whereas the gel-like fraction was not apparently modified, the volume of the liquid vitreous fraction (LVF) was much higher in the myopic eyes. Biochemical and proteome analysis of the LVF revealed several modifications including a marked decrease of potassium, sodium and chloride, of proteins involved in ocular tissue homeostasis and repair as well as of ADP-ribosylation factor 4 (ARF4), a protein possibly involved in LRP2 trafficking. A small number of proteins, mainly comprising known LRP2 ligands or proteins of the inflammatory response, were over expressed in the mutants. Moreover the morphology of the LRP2-deficient retinal pigment epithelium (RPE) cells was affected and the expression of ARF4 as well as of proteins involved in degradative endocytosis was strongly reduced. Our results support the idea that impairment of the RPE structure and most likely endocytic function may contribute to the vitreal modifications and pathogenesis of HM.

Brain proteomic modifications associated to protective effect of grape extract in a murine model of obesity.

In recent years, the obesity epidemic has developed into a major health crisis worldwide. With current treatments limited to expensive, high-risk surgery and minimally efficacious pharmacotherapy, new therapeutic options are urgently needed to fight against this alarming trend. Though brain dysfunction has been studied linked to high fat diet (HFD) and grape seed and skin extract (GSSE) correction, the proteomic modifications linking the two effects on brain lipotoxicity are not well understood. To this end rats were exposed for 8 weeks to HFD treatment, to GSSE (500mg/kg BW) and to binary mixture of HFD and GSSE to gain insight into the potential pathways altered with metabolic disease and the protection afforded by GSSE. Significant modifications of brain proteins were detected using mass spectrometry-based differential proteomics. These proteins were mainly related to oxidative stress, glycolysis and calcium signaling. Additionally, proteins involved in the cytoskeleton were also affected by HFD treatment. Interestingly, whether up- or down regulated protein abundances, GSSE corrected most of the disturbances of HFD treatment. These findings provide impetus for future therapeutic investigation on GSSE against other metabolic disorders.

Proteomics dedicated to biofilmology: What have we learned from a decade of research?

Advances in proteomics techniques over the past decade, closely integrated with genomic and physicochemical approach, have played a great role in developing knowledge of the biofilm lifestyle of bacteria. Despite bacterial proteome versatility, many studies have demonstrated the ability of proteomics approaches to elucidating the biofilm phenotype. Though these investigations have been largely used for biofilm studies in the last decades, they represent, however, a very low percentage of proteomics works performed up to now. Such approaches have offered new targets for combating microbial biofilms by providing a comprehensive quantitative and qualitative overview of their protein cell content. Herein, we summarized the state of the art in knowledge about biofilm physiology after one decade of proteomic analysis. In a second part, we highlighted missing research tracks for the next decade, emphasizing the emergence of posttranslational modifications in proteomic studies stemming from recent advances in mass spectrometry-based proteomics.

Proteomic profiling of lysine acetylation in Pseudomonas aeruginosa reveals the diversity of acetylated proteins.

Protein lysine acetylation is a reversible and highly regulated post-translational modification with the well demonstrated physiological relevance in eukaryotes. Recently, its important role in the regulation of metabolic processes in bacteria was highlighted. Here, we reported the lysine acetylproteome of Pseudomonas aeruginosa using a proteomic approach. We identified 430 unique peptides corresponding to 320 acetylated proteins. In addition to the proteins involved in various metabolic pathways, several enzymes contributing to the lipopolysaccharides biosynthesis were characterized as acetylated. This data set illustrated the abundance and the diversity of acetylated lysine proteins in P. aeruginosa and opens opportunities to explore the role of the acetylation in the bacterial physiology.

Determination of Multimodal Isotopic Distributions: The Case of a (15)N Labeled Protein Produced into Hairy Roots.

Isotopic labeling is widely used in various fields like proteomics, metabolomics, fluxomics, as well as in NMR structural studies, but it requires an efficient determination of the isotopic enrichment. Mass spectrometry is the method of choice for such analysis. However, when complex expression systems like hairy roots are used for production, multiple populations of labeled proteins may be obtained. If the isotopic incorporation determination is actually well-known for unimodal distributions, the multimodal distributions have scarcely been investigated. Actually, only a few approaches allow the determination of the different labeled population proportions from multimodal distributions. Furthermore, they cannot be used when the number of the populations and their respective isotope ratios are unknown. The present study implements a new strategy to measure the (15)N labeled populations inside a multimodal distribution knowing only the peptide sequence and peak intensities from mass spectrometry analyses. Noteworthy, it could be applied to other elements, like carbon and hydrogen, and extended to a larger range of biomolecules.

Characterization of new outer membrane proteins of Pseudomonas aeruginosa using a combinatorial peptide ligand library.

Most often, the use of ProteoMiner beads has been restricted to human serum proteins for the normalization of major proteins, such as albumin. However, there are other situations of interest in which the presence of major proteins would quench the signals of low abundance polypeptides. We propose the use of these beads for investigating the envelope of the gram-negative bacterium Pseudomonas aeruginosa. Initially, we performed comparative 2D electrophoresis to qualitatively evaluate the incidence of the normalization stage. This demonstrated a significant reduction of the major membrane proteins. Thereafter, using shotgun analysis, the same protein extract was targeted by using combinatorial peptide ligand library capture. This treatment yielded 154 additional outer membrane proteins (OMPs) uncovered by the study of the crude sample.

Characterization of endophytic Bacillus strains from tomato plants (Lycopersicon esculentum) displaying antifungal activity against Botrytis cinerea Pers.

Eighty endophytic bacteria were isolated from healthy tissues of roots, stems, leaves and fruits of tomato plants (Lycopersicon esculentum). Four strains, named BL1, BT5, BR8 and BF11 were selected for their antagonism against Botrytis cinerea, a phytopathogenic fungus responsible of gray mold in several important crops, with growth inhibitory activity ranging from 27 to 53%. Morphological, biochemical, and molecular parameters as 16S rDNA sequencing demonstrated that the selected bacterial strains were related to Bacillus species which are known to produce and secrete a lot of lipopeptides with strong inhibitory effect against pathogen mycelial growth. Electrospray mass spectrometry analysis showed that these strains produced heterogeneous mixture of antibiotics belonging to fengycin and surfactin for BL1 and BT5, to iturin and surfactin for BR8, to bacillomycin D, fengycin and surfactin for BF11. Furthermore, these bacteria exhibited biocontrol potential by reducing the disease severity when tested on detached leaflets. Based on their antifungal activity against Botrytis cinerea, these strains could be used for biological control of plant diseases.

Extracellular Ser/Thr/Tyr phosphorylated proteins of Pseudomonas aeruginosa PA14 strain.

Protein phosphorylation on serine, threonine, and tyrosine is known to be involved in a wide variety of cellular processes, signal transduction, and bacterial virulence. We characterized, for the first time, the extracellular phosphoproteins of the Pseudomonas aeruginosa PA14 strain. We identified 28 phosphoproteins (59 phosphosites) including enzymes, with various phosphorylation sites, known as potent secreted virulence factors in P. aeruginosa. The high phosphorylation level of these virulence factors might reflect a relationship between Ser/Thr/Tyr phosphorylation and virulence.

Design and synthesis of epicocconone analogues with improved fluorescence properties.

Epicocconone is a natural latent fluorophore that is widely used in biotechnology because of its large Stokes shift and lack of fluorescence in its unconjugated state. However, the low photostability and quantum yields of epicocconone have limited its wider use, and in the absence of a total synthesis, this limitation has been a long-standing problem. Here we report a general strategy for the synthesis of epicocconone analogues that relies on a 2-iodoxybenzoic acid-mediated dearomatization and on the replacement of the triene tail of the natural product by an aromatic ring. This design element is general and the synthesis is straightforward, providing ready access to libraries of polyfunctional fluorophores with long Stokes shifts based on the epicocconone core. Our structural modifications resulted in analogues with increased photostability and quantum yields compared with the natural product. Staining proteomic gels with these new analogues showed significant lowering of the detection limit and a 30% increase in the number of low-abundance proteins detected. These epiccoconone analogues will substantially improve the discovery rate of biomarker needles in the proteomic haystack.

Growth retardation, reduced invasiveness, and impaired colistin-mediated cell death associated with colistin resistance development in Acinetobacter baumannii.

Two colistin-susceptible/colistin-resistant (Col(s)/Col(r)) pairs of Acinetobacter baumannii strains assigned to international clone 2, which is prevalent worldwide, were sequentially recovered from two patients after prolonged colistin administration. Compared with the respective Col(s) isolates (Ab248 and Ab299, both having a colistin MIC of 0.5 µg/ml), both Col(r) isolates (Ab249 and Ab347, with colistin MICs of 128 and 32 µg/ml, respectively) significantly overexpressed pmrCAB genes, had single-amino-acid shifts in the PmrB protein, and exhibited significantly slower growth. The Col(r) isolate Ab347, tested by proteomic analysis in comparison with its Col(s) counterpart Ab299, underexpressed the proteins CsuA/B and C from the csu operon (which is necessary for biofilm formation). This isolate also underexpressed aconitase B and different enzymes involved in the oxidative stress response (KatE catalase, superoxide dismutase, and alkyl hydroperoxide reductase), suggesting a reduced response to reactive oxygen species (ROS) and, consequently, impaired colistin-mediated cell death through hydroxyl radical production. Col(s) isolates that were indistinguishable by macrorestriction analysis from Ab299 caused six sequential bloodstream infections, and isolates indistinguishable from Ab248 caused severe soft tissue infection, while Col(r) isolates indistinguishable from Ab347 and Ab249 were mainly colonizers. In particular, a Col(s) isolate identical to Ab299 was still invading the bloodstream 90 days after the colonization of this patient by Col(r) isolates. These observations indicate considerably lower invasiveness of A. baumannii clinical isolates following the development of colistin resistance.

Overexpression of MHC class I in muscle of lymphocyte-deficient mice causes a severe myopathy with induction of the unfolded protein response.

Muscle fibers do not normally express major histocompatibility complex class I (MHC-I) molecules, and their reexpression is a hallmark of inflammatory myopathies. It has been shown in mice that overexpression of MHC-I induces a poorly inflammatory myositis accompanied by the unfolded protein response (UPR), but it is unclear whether it is attributable to T-cell-mediated MHC-I-dependent immune responses or to MHC-I forced expression per se. Indeed, besides presenting antigenic peptides to CD8(+) T cells, MHC-I may also possibly exert nonimmunologic, yet poorly understood pathogenic effects. Thus, we investigated the pathogenicity of MHC-I expression in muscle independently of its immune functions. HT transgenic mice that conditionally overexpress H-2K(b) in muscle were bred to an immunodeficient Rag2(-/-) background. The muscle proteome was analyzed by label-free high-resolution protein quantitation and Western blot. Despite the absence of adaptive immunity, HT Rag2(-/-) mice developed a very severe myopathy associated with the cytoplasmic accumulation of H-2K(b) molecules. The UPR was manifest by up-regulation of characteristic proteins. In humans, we found that HLA class I molecules not only were expressed at the sarcolemma but also could accumulate intracellularly in some patients with inclusion body myositis. Accordingly, the UPR was triggered as a function of the degree of HLA accumulation in myofibers. Hence, reexpression of MHC-I in normally negative myofibers exerts pathogenic effects independently of its immune function.

Potential of liquid-isoelectric-focusing protein fractionation to improve phosphoprotein characterization of Pseudomonas aeruginosa PA14.

Protein phosphorylation on serine, threonine, and tyrosine is known to be involved in a wide variety of cellular processes and signal transduction in bacteria. Bacterial-proteome analysis is required to determine which proteins have been conditionally expressed and whether any post-translational modifications are present. One of the greatest challenges of proteome analysis is the fractionation of these complex protein mixtures to detect low-abundance phosphoproteins. Liquid-phase isoelectric focusing (IEF) is a promising analytical tool in proteomics, but as far as we are aware no work has studied the reproducibility of this approach. In this study, we investigated the phosphoproteome of Pseudomonas aeruginosa strain PA14. We first tested in-solution IEF protein fractionation, and then used this technique to fractionate the proteins in the complex mixture. Next, phosphopeptides were enriched with titanium dioxide and analyzed by high-resolution, high-accuracy liquid chromatography-mass spectrometry. With this approach, we succeeded in characterizing 73 unique phosphorylated peptides belonging to 63 proteins. Interestingly, we observed a higher percentage of modified tyrosine, revealing the importance of this phosphorylated residue in bacteria.

Proteomic regulation during Legionella pneumophila biofilm development: decrease of virulence factors and enhancement of response to oxidative stress.

Legionella pneumophila (L. pneumophila) is a Gram-negative bacterium, which can be found worldwide in aquatic environments. It tends to persist because it is often protected within biofilms or amoebae. L. pneumophila biofilms have a major impact on water systems, making the understanding of the bacterial physiological adaptation in biofilms a fundamental step towards their eradication. In this study, we report for the first time the influence of the biofilm mode of growth on the proteome of L. pneumophila. We compared the protein patterns of microorganisms grown as suspensions, cultured as colonies on agar plates or recovered with biofilms formed on stainless steel coupons. Statistical analyses of the protein expression data set confirmed the biofilm phenotype specificity which had been previously observed. It also identified dozens of proteins whose abundance was modified in biofilms. Proteins corresponding to virulence factors (macrophage infectivity potentiator protein, secreted proteases) were largely repressed in adherent cells. In contrast, a peptidoglycan-associated lipoprotein (Lpg2043) and a peroxynitrite reductase (Lpg2965) were accumulated by biofilm cells. Remarkably, hypothetical proteins, that appear to be unique to the Legionella genus (Lpg0563, Lpg1111 and Lpg1809), were over-expressed by sessile bacteria.

IFNγ causes mitochondrial dysfunction and oxidative stress in myositis.

Idiopathic inflammatory myopathies (IIMs) are severe autoimmune diseases with poorly understood pathogenesis and unmet medical needs. Here, we examine the role of interferon γ (IFNγ) using NOD female mice deficient in the inducible T cell co-stimulator (Icos), which have previously been shown to develop spontaneous IFNγ-driven myositis mimicking human disease. Using muscle proteomic and spatial transcriptomic analyses we reveal profound myofiber metabolic dysregulation in these mice. In addition, we report muscle mitochondrial abnormalities and oxidative stress in diseased mice. Supporting a pathogenic role for oxidative stress, treatment with a reactive oxygen species (ROS) buffer compound alleviated myositis, preserved muscle mitochondrial ultrastructure and respiration, and reduced inflammation. Mitochondrial anomalies and oxidative stress were diminished following anti-IFNγ treatment. Further transcriptomic analysis in IIMs patients and human myoblast in vitro studies supported the link between IFNγ and mitochondrial dysfunction observed in mice. These results suggest that mitochondrial dysfunction, ROS and inflammation are interconnected in a self-maintenance loop, opening perspectives for mitochondria therapy and/or ROS targeting drugs in myositis.

Extracellular vesicles from type-2 macrophages increase the survival of chronic lymphocytic leukemia cells ex vivo.

The resistance of Chronic Lymphocytic Leukemia (CLL) B-cells to cell death is mainly attributed to interactions within their microenvironment, where they interact with various types of cells. Within this microenvironment, CLL-B-cells produce and bind cytokines, growth factors, and extracellular vesicles (EVs). In the present study, EVs purified from nurse-like cells and M2-polarized THP1 cell (M2-THP1) cultures were added to CLL-B-cells cultures. EVs were rapidly internalized by B-cells, leading to a decrease in apoptosis (P = 0.0162 and 0.0469, respectively) and an increased proliferation (P = 0.0335 and 0.0109). Additionally, they induced an increase in the resistance of CLL-B-cells to Ibrutinib, the Bruton kinase inhibitor in vitro (P = 0.0344). A transcriptomic analysis showed an increase in the expression of anti-apoptotic gene BCL-2 (P = 0.0286) but not MCL-1 and an increase in the expression of proliferation-inducing gene APRIL (P = 0.0286) following treatment with EVs. Meanwhile, an analysis of apoptotic protein markers revealed increased amounts of IGFBP-2 (P = 0.0338), CD40 (P = 0.0338), p53 (P = 0.0219) and BCL-2 (P = 0.0338). Finally, exploration of EVs protein content by mass spectrometry revealed they carry various proteins involved in known oncogenic pathways and the RNAseq analysis of CLL-B-cells treated or not with NLCs EVs show various differentially expressed genes.

BacA: a possible regulator that contributes to the biofilm formation of Pseudomonas aeruginosa.

Previously, we pointed out in P. aeruginosa PAO1 biofilm cells the accumulation of a hypothetical protein named PA3731 and showed that the deletion of the corresponding gene impacted its biofilm formation capacity. PA3731 belongs to a cluster of 4 genes (pa3732 to pa3729) that we named bac for "Biofilm Associated Cluster." The present study focuses on the PA14_16140 protein, i.e., the PA3732 (BacA) homolog in the PA14 strain. The role of BacA in rhamnolipid secretion, biofilm formation and virulence, was confirmed by phenotypic experiments with a bacA mutant. Additional investigations allow to advance that the bac system involves in fact 6 genes organized in operon, i.e., bacA to bacF. At a molecular level, quantitative proteomic studies revealed an accumulation of the BAC cognate partners by the bacA sessile mutant, suggesting a negative control of BacA toward the bac operon. Finally, a first crystallographic structure of BacA was obtained revealing a structure homologous to chaperones or/and regulatory proteins.